valuable insights into both the strengths and weaknesses of the proposed research. With regard to strengths, Reviewer #1 said, "This application contains an innovative plan. Byinvestigating a link between the current hypotheses, insteadof attempting toprove one while debunking the other, the author shows forward thinking." "This application is for a body of work that is likely to make amajor impact onALS research." Reviewer#2 wrote: "The plan is reasonable and logical. The investigator's proposalis well written andprovides anice conceptual framework for his proposedstudies." "Theproposal...addresses an important issue relevant to all neurodegenerative diseases-altered conformation, in this case, the altered conformation off ALS SOD proteins as the cause of motor neuron degeneration." The general positive tone of these comments was echoed by Reviewer #3: "There are interesting experiments that address important questions, and therefore, are worth carrying out. Dr. Hart has contributed significantly to our understanding of mutant SOD1 aggregation andhepossesses all the technical expertise to bring the proposal to a successful conclusion." "The proposal uses establishedmethods in structural biology and biochemistry to answer important questions in theALS field. Theexperimental ideas, Aim 1inparticular, areinnovative." There were also some criticisms of the application. The Summary of Discussion stated, "The principal concern with Project 2 wasthe level of work proposed. In light of the labor-intensive nature of the technique tobe used, the project appearedoverly ambitious. It wasfelt that while the approach wasexcellent, it may be more usefulto limit the investigations to those mutants most likely to provide useful information." Reviewer#2 agreed with this and wrote, "Particularly, the number of fALS mutant proteins generated (and in modified versions) along with the proposal to test a large number of them in vivo with Dr. Borchelt's group may represent a body of work that is redundant and too ambitious." "[The applicant should]...reduce the number of proposedexperiments making SODtransgenic animals." A different criticism came from Reviewer #1 who said, "The applicant argues that a lack of metals maybea preliminary step in aggregation through monomerization of the pathogenic SOD. This hypothesis is not upheldby the crystal structures of the pathogenic SODillustratedin the preliminary evidence, which cleariy show that the building block for either the amyloid-like or pore-like filaments are dimers." Finally, there were some specific questions about technical issues that need to be addressed in the revised application. The responses to the reviewer's comments have been grouped into three major categories in this introduction: (1) The ambitious nature of the original proposal; (2) Monomers versus dimers in pathogenic SOD1 aggregation; and (3) Specific technical criticisms. The original proposal was written in Anal font. To facilitate review of the revised application, those portions that have undergone major modification are shown in Times font, as in this paragraph. In addition, small editorial alterations have been made to improve readability. These are not highlighted. 1. Theambitious nature of the originalproposal: The principal concern with the proposed researchwas that it was too ambitious. The reviewers felt that too many pathogenic mutants were to be tested in vivo (that is, in mice) in collaboration with Dr. Borchelt (Project 3) and that in vitro and cell culture studies should be performed first to indicate which mutants are interesting enough to merit moving into transgenic animals. We agree with this criticism and the revised application has been modified in the Research Design and Methods section to clearly indicate that *only* the most interesting mutants as defined by these initial studies will be moved into the transgenic animals. In terms of producing mutant proteins for biophysical studies, however, the Hart laboratory has already cloned and is expressing most of the SOD1 constructs needed for in vitro work (see Table 1 in the revised application) and thus, the proposedin vitro biophysical studies that are the focus of this project (Project 2) are likely to be successful. A different criticism regarding the scope of the proposal came from Reviewer #3 who wrote about the experiments outlined in Aim 4, "While this is a valuable research goal, this aimseems out ofplace in this proposal- especially considering its inclusion in aPPG in whichDr. Levine is not a contributor." Given the consensus that the original proposal was too ambitious, and because Aim 4 differsfrom Aims 1-3 in both focus and methodology, Aim 4 will be eliminated from this research project and will serve as the basis for a separate series of studies. PHS 398/2590 (Rev.OS/01) Page 121 Continuation Format Page